We have successfully integrated MSe acquisitions into our proteomics experiments and have closely worked with bioinformatics group to validate our results on several projects. 1. Method Development. The implementation of label-free approaches for quantitative proteomics studies is being implemented. This methodology involves data-independent analyses (MSe) and is being evaluated to determine if this MS approach is better for protein quantitation studies than data dependent (DDA) methods. In addition, an online 2D LC method suitable for the identification of proteins from complex samples is being optimized. MSe can rapidly acquire accurate mass precursor and fragment ion information while simultaneously obtaining fairly accurate quantitative profiles from every detectable component in the sample, in theory. 2. Proteomic Studies Involving Myositis. A differential proteomics project comparing the quantitation of proteins from sera of healthy individuals to individuals diagnosed with a rheumatic disease is underway. This work is in collaboration with F. Miller (EAG) as part of the EAGs study of families with twins or siblings discordant for systemic rheumatic disorders. The goal of this project is to determine whether a protein(s) can be identified that would allow for the early diagnosis, prognosis, and treatment of these diseases. Sera samples have been depleted and processed and are awaiting LC/MSe analysis. 3. Proteomic Studies of CSF. As an extension of the gene expression studies of COS (carbonyl sulfide) treated rats (R. Sills, MCP), a protein expression study on CSF of rats as a result of COS treatment was investigated. The samples were collected from 3 independent treatments including 1 day, 2 day, and 3 day exposures. Total protein concentration was determined. Additionally, the FFPE brain tissues were evaluated (formalin-fixed paraffin-embedded). FFPE is a common tissue preservation technique and is routinely performed at NIEHS. We have found that refinements of published protocols are needed to evaluate the suitability of LC-MSe for the analysis of extracted FFPE tissue proteins from FFPE blocks. Initial experiments using FFPE brain tissues of six rats compared extraction efficacies of proteins from FFPE tissues using home-made and published protocols. The best results were obtained when the tissue sections were deparaffinized in xylene and rehydrated through a series of alcohol treatments and when the protein extractions were carried out using an acid labile surfactant. 4. Proteomic Studies following Environmental Exposure. A quantitative serum proteomics study of serum from healthy individuals following time spent in an isolation chamber under conditions similar to a space-like life support system was designed to address potential long-term effects of space exploration. Plasma samples were taken at six different time points throughout a 103 day isolation period from six individuals. The samples were depleted of the 14 most abundant proteins, digested, and analyzed by LC/MSe. A manuscript reporting these results is being finalized. 5. Proteomic Studies of Yeast. To get an understanding of what can be observed from the yeast proteome (P. Blackshear LST), different protein extraction buffers were used and the resulting 2DLC/MSe analyses were performed.